Localization of 1,25-(OH)2D3-responsive alkaline phosphatase in osteoblast-like cells (ROS 17/2.8, MG 63, and MC 3T3) and growth cartilage cells in culture

B. D. Boyan, Zvi Schwartz, L. F. Bonewald, L. D. Swain

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Abstract

Previous studies have shown 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-responsive alkaline phosphatase in cultured growth zone cartilage chondrocytes is localized in extracellular matrix vesicles (MV). Since osteoblast-like cells also have 1,25-(OH)2D3-responsive alkaline phosphatase, this study determined whether the 1,25-(OH)2D3-responsive enzyme activity is localized to MV produced by these cells as well. Osteoblast-like cells from rat (ROS 17/2.8), mouse (MC 3T3), human (MG 63), and rat growth zone cartilage were cultured in Dulbecco's modified Eagle's medium containing 10-7-10-12 M 1,25-(OH)2D3. Alkaline phosphatase total activity and specific activity were measured in the cell layer, MV, and plasma membrane (PM) fractions. MV and PM purity were verified by electron microscopy and MV alkaline phosphatase specific activity compared to PM (MV versus PM: ROS 17/2.8 6 x; MG 63, 5.5 x; MC 3T3, 33 x; GC, 2 x). There was a dose-dependent stimulation of MV alkaline phosphatase (5- to 15-fold increase at 10-7-10-9 M) in all cell types in response to the 1,25-(OH)2D3. The PM enzyme was stimulated in a parallel fashion in the osteoblast cultures. No effect of 1,25-(OH)2D3 was observed in growth cartilage PM. Although MV accounted for < 20% of the total activity they contributed 50% of the increase in alkaline phosphatase activity in the cell layer in response to 1,25-(OH)2D3 and MV specific activity was enriched 10 times over that of the cell layer. These are common features of MV produced by cells which calcify their matrix and suggest that hormonal regulation of MV enzymes may be important in primary calcification.

Original languageEnglish (US)
Pages (from-to)11879-11886
Number of pages8
JournalJournal of Biological Chemistry
Volume264
Issue number20
StatePublished - 1989

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Osteoblasts
Cartilage
Cell membranes
Alkaline Phosphatase
Cell Culture Techniques
Cell Membrane
Growth
Cells
Rats
Enzymes
Calcitriol
Enzyme activity
Cell culture
Eagles
Electron microscopy
Chondrocytes
Extracellular Matrix
Electron Microscopy

ASJC Scopus subject areas

  • Biochemistry

Cite this

Localization of 1,25-(OH)2D3-responsive alkaline phosphatase in osteoblast-like cells (ROS 17/2.8, MG 63, and MC 3T3) and growth cartilage cells in culture. / Boyan, B. D.; Schwartz, Zvi; Bonewald, L. F.; Swain, L. D.

In: Journal of Biological Chemistry, Vol. 264, No. 20, 1989, p. 11879-11886.

Research output: Contribution to journalArticle

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title = "Localization of 1,25-(OH)2D3-responsive alkaline phosphatase in osteoblast-like cells (ROS 17/2.8, MG 63, and MC 3T3) and growth cartilage cells in culture",
abstract = "Previous studies have shown 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-responsive alkaline phosphatase in cultured growth zone cartilage chondrocytes is localized in extracellular matrix vesicles (MV). Since osteoblast-like cells also have 1,25-(OH)2D3-responsive alkaline phosphatase, this study determined whether the 1,25-(OH)2D3-responsive enzyme activity is localized to MV produced by these cells as well. Osteoblast-like cells from rat (ROS 17/2.8), mouse (MC 3T3), human (MG 63), and rat growth zone cartilage were cultured in Dulbecco's modified Eagle's medium containing 10-7-10-12 M 1,25-(OH)2D3. Alkaline phosphatase total activity and specific activity were measured in the cell layer, MV, and plasma membrane (PM) fractions. MV and PM purity were verified by electron microscopy and MV alkaline phosphatase specific activity compared to PM (MV versus PM: ROS 17/2.8 6 x; MG 63, 5.5 x; MC 3T3, 33 x; GC, 2 x). There was a dose-dependent stimulation of MV alkaline phosphatase (5- to 15-fold increase at 10-7-10-9 M) in all cell types in response to the 1,25-(OH)2D3. The PM enzyme was stimulated in a parallel fashion in the osteoblast cultures. No effect of 1,25-(OH)2D3 was observed in growth cartilage PM. Although MV accounted for < 20{\%} of the total activity they contributed 50{\%} of the increase in alkaline phosphatase activity in the cell layer in response to 1,25-(OH)2D3 and MV specific activity was enriched 10 times over that of the cell layer. These are common features of MV produced by cells which calcify their matrix and suggest that hormonal regulation of MV enzymes may be important in primary calcification.",
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